Download Transfection - Biomanufacturing.org

Transfection
Key words: Transient transfection, Stable transfection, transfection
methods, vector, plasmid, origin of replication, reporter gene/ protein, cloning
site, promoter and enhancer, signal peptide, polyadenylation signal.
• Transfection-Transfer of non-viral genetic material
into eukaryotic cells.
• Infection/ Transduction- Transfer of viral genetic
material into cells.
• Transformation- Transfer of genetic material into
bacterial and plant cells.
Transfection
• The goal of transfection is to express a
particular gene in the host cell.
• Used to Study : Gene expression regulations,
Protein function, Gene silencing, Gene therapy
and more.
Transient Transfection
• When the introduced DNA molecule is not
inserted into the host genome.
•The DNA is eventually lost during mitosis.
•Transiently transfected cells are assayed 48-72
hours after transfection.
Stable Transfection
• Occasionally the introduced DNA is inserted
into the host genome. The DNA becomes part
of the host genome and is passed to the next
generations.
• The cells that are stably transfected need to be
selected for and separated from the transient or
non-transfected cells. The stably transfected
cells are normally selected for about 48 hours
post transfection.
Transfection Methods
• Negatively charged DNA
interacts with the cationic
molecules or polymers and
is taken into the cell by
endocytosis.
• Examples:
Calcium Phosphate
cationic liposomes
Other Transfection Methods:
Nanoparticles
• Nanoparticls are coated with DNA and directly shot
into the nucleus.
Microinjection
• Direct injection into individual cells by a very thin
needle.
Electroporation
• The electric potential across the membrane drives
DNA across the membrane through the pores.
• Charged ions and DNA flow through the pores, then
the cell membrane discharges and the pores close and
the DNA molecules are inside the cell.
Vectors
• Carrier DNA sequences that are able to
enter the host cells
• Able to replicate independently in the host
cell
• Able to express genes in the host cell
• Plasmids, artificial chromosomes and
viruses are commonly used as vectors.
• Non-viral vectors rely on cell division for
transfer of DNA into the nucleus.
Plasmids:
Extra-chromosomal DNA molecules found in
bacteria.
•Double stranded DNA
•Circular
•Carry genes with variety of functions
Plasmids
Bacterial DNA
Plasmids are genetically engineered to be used
as vectors that can carry foreign DNA.
Plasmids that are commonly used for biotechnology
have the following features:
• Small size (1-500 kb)
• Origin of replication- Can replicate in the host cell
and make high copy numbers
• Selectable marker gene
• Cloning site
Origin of Replication
• A sequence of DNA where replication is initiated.
• Plasmids are often amplified in bacteria and then used
for transfection in other cell types. Therefore they
posses two different origins of replication suitable for
both cell types.
• Some origins of replications allow more efficient
replications and yield high copy number of plasmids.
• High copy number origins are preferred since more
plasmids are replicated in shorter time.
Cloning site
Refers to multiple restriction enzyme
sites on the plasmid’s sequence where
foreign DNA can be inserted.
Reporter Gene (Selectable marker)
• A reporter gene used to select for the
transfected cells and separate them from nontransfected cells.
• Antibiotic resistant genes or genes coding for
fluorescent proteins are commonly used as
selectable markers.
In addition plasmids may be genetically
engineered to contain specific DNA sequences
such as:
• Enhancer and promoter sequences
• Signal peptides
• Polyadenylation sequence
Enhancer and promoter sequences
• Regulatory sequences where transcription factors
and regulatory proteins bind and control expression of
a specific gene.
• Enhancer and promoter sequences of different genes
may be specific to an organism and may be active only
in specific tissues.
• Plasmids are engineered with appropriate regulatory
sequences that are inserted upstream of the gene of
interest (5’ to the RE sites) specific to an organism and
may be specific to a tissue.
Signal peptides
• A short peptide sequence on every protein that
used to interact with transport systems in the cell
to direct the protein to the correct cellular
location.
• Signal peptides are often cleaved off once the
final destination has been reached.
• A plasmid may be engineered to carry a signal
sequence to direct the protein product of the
inserted gene to a specific location inside the cell
or direct it to be secreted outside.
Polyadenylation sequence
• A specific sequence recognized by the
polyadenylation machinery which adds a stretch
of Adenines to the tail end (3’) of the RNA
molecule.
• RNA molecules that are polyadenylated are said
to be matured (mRNA) and are more stable.
• Polyadenylation sequence may be inserted 3’ to
the cloning site to be used for polyadenylation of
the RNA product.
pEGFP‐Actin VECTOR
From Clontech
• pUC Ori- Origin of replication for propagation in bacteria.
• SV40 Ori- Origin of replication for replication in
mammalian cells. Taken from the SV40 virus.
• pCMV IE- Cytomegalovirus promoter for gene expression
in mammalian cells
• pSV40- SV40 promoter for gene expression in mammalian
cells
• P- Bacterial promoter for gene expression in Ecoli bacteria
• SV40 Poly A and HSV TK Poly A- Polyadenylation and
stabalization of the mRNA expressed in mammalian cells.
• Neo/ Kan (r)- Selectable genes that encode an enzyme to
make both bacterial and mammalian cells resistant to
genticin, neomycin and kanamycin. (These antibiotics kill cells by
interfering with protein synthesis.)